The structure and biological activity of three oncogene-encoded proteins were studied. Proteins encoded by two oncogens, v-fes and v-abl, are tyrosine specific protein kinases which phosphorylate both themselves and other substrates. The phosphorylation sites on P120gag-abl were identified on three tryptic peptides and found to be 1, 6, and 7 residues distal to their tryptic cleavage sites. The sequence of one of these peptides was deduced from the corresponding DNA sequence of the human c-abl gene and the peptide was chemically synthesized. Immunoprecipitated P120gag-abl efficiently phosphorylated this peptide. Tryptic peptides prepared from v-fes gene-encoded polyproteins were found to have a similar phosphorylation site at a position seven residues distal to the cleavage site. A phosphoglycoprotein of 150,000 daltons (P150) was identified as a substrate for the v-abl- and v-fes-encoded protein kinases; it was found to be a serine-specific protein kinase. P150 was shown to be distinct from each of several previously demonstrated substrates for tyrosine-specific protein kinases, including vinculin, several glycolytic enzymes and P39. Its phosphorylation may be involved in the regulation of cellular events resulting in expression of the transformed phenotype. Expression of this protein was restricted to epithelial cells and carcinomas, suggesting that it may be a useful tumor marker. Cells transformed by both the v-abl and v-fes genes were found to secrete a tumor growth factor that is structurally related to epidermal growth factor. This factor, in concert with a relatively ubiquitous potentiating factor, stimulated the growth of cells in soft agar. A third oncogene studied, v-raf, was found to encode two proteins of 75,000 and 90,000 daltons that are not tyrosine-specific protein kinases. v-raf-transformed cells do not exhibit elevated levels of phosphotyrosine. The v-raf gene was found to differ from each of a number of other oncogenes examined and, thus, is thought to represent a unique new oncogene.